Severe asthma remains a debilitating and difficult to control disease. Notwithstanding the basic understanding that asthma is initiated and perpetuated by a strong inflammatory component, in many patients, the disease manifests in a severe form that is unresponsive to treatment by corticosteroids (CS). However, asthma in milder forms generally responds to standard steroid therapy. Our preliminary data presented in two human- based projects suggest that severe asthma cannot be explained simply by an overzealous Th2 response and eosinophilic airway inflammation. The overall hypothesis for this P01 application is that the pathophysiology of steroid refractory severe asthma involves cooperativity between a heightened Th1 (IFN-?) immune response often accompanied by increased IL-27 levels, residual Type 2 responses plus a deficient IL-10 response. This atypical immune response, not appreciated heretofore, induces specific downstream effects on airway epithelial cells such as profound nitro-oxidative stress, which perpetuate the inflammatory response resulting in chronic disease. Our hypothesis will be addressed in 2 Projects. Project 1 will establish the atypical immune fingerprint of CS-refractory severe asthma (SA) as compared to milder asthma using immunological and RNA-sequencing (RNA-Seq) approaches. A newly established mouse model of SA will be used to understand the role of immune pathways in inducing airway oxidative stress. The mechanisms by which the combination of IL- 27 and IFN-? causes glucocorticoid receptor (GR) dysfunction will be elucidated. RNA-sequencing (RNA-seq) approaches in conjunction with bioinformatic tools will be used to derive a gene interactome of SA. Project 2 will identify the molecular phenotypes induced by the intersection of Type 1 and 2 immune pathways, and IL-27 on primary airway epithelial cells using biased and unbiased (RNA-Seq) approaches. The role of nitro-oxidative stress in inducing these phenotypes will be determined. Also, whether these cytokine combinations induce CS- unresponsiveness in epithelial cells will be studied along with the possibility of improving the response by IL-10. Importantly both projects will examine the heightened STAT1 activation with low/no STAT3 activation observed in immune and epithelial cells, this altered balance being detrimental for both immune regulation and epithelial cell function. Synergistic interactions among projects will be afforded by support from the Administrative Core, to coordinate the activities of the P01 at all levels, the Human Biological Sampling and Immunocytometry Core and, the RNA-Seq and Bioinformatics Core. Thus, using cutting edge immunological, cellular and RNA sequencing techniques, the proposal will establish a new paradigm for severe asthma based on which novel therapeutics could be developed in the future.